LEAP Testing Service

WESTERN BLOT ANALYSIS

 I.  Scope
This Western blot procedure qualitatively assesses the molecular weight distribution of antigenic protein in latex extracts by separating the proteins on an SDS-PAGE, transferring them to nitrocellulose and then reacting the blot using anti-latex antibody.

II.  Procedure

1. Dialyze (1,000 MWCO) 6 ml of each sample against two exchanges (at least 3 hours each) with H₂O (6 ml to 250 ml for each exchange).
2. Lyophilize 5 ml of each dialyzed sample overnight to concentrates protein.
3. Dissolve each sample in 100 ul of reducing SDS-PAGE sample buffer and boil samples for 3minutes.
4. Load 45 µl onto a 15% SDS-PAGE gel.
5. Run until dye front has run to the bottom of the gel. Stop gel by turning off current and removing leads. For Hoefer Minigel set up running time is approximately 2 hours for the dye front to reach the bottom when run at a constant voltage of 100V.
6. Remove spacers from gel and gently pry plates apart. 
7. Set up transfer sandwich in container with transfer buffer. Set up sandwich as follows: Cassette frame with black side down, soaked scotch brite pad, hours pieces precut blotting paper, SDS-gel, nitrocellulose membrane, 2X blotting paper, scotch brite pad and clear cassette frame. Close the frame and secure with top clasp (nick a bottom corner to help with gel orientation). Transfer buffer is Towbin's. Nitrocellulose should be prewetted in deionized distilled water then Towbin's buffer. The sandwich should be free of air bubbles.
8. Place sandwich into slot of stand in the transfer tank, which is half filled with Towbin's buffer. Place black side of sandwich next to black side of transfer stand. Fill buffer tank with Towbin's buffer. Do not overfill tank as buffer will expand as it warms. Buffer level should be high enough that gel is submerged.
9. Place a stir bar in the tank and place on a magnetic stir plate. Secure lid and plug leads into power supply. Stirring buffer assures constant temperature as well as pH. Transfer proteins with a constant current of 150 mA for 3 hours Transfer can proceed overnight at a lower current.
10. When the transfer is complete, turn off power and remove blot from apparatus. Remove membrane and place in glass dish with Ponceau S stain. Staining will give an indication of transfer efficiency.
11. Block membrane with a solution of 3% BSA or 5% non-fat milk protein in PBS. Block for 1 hour or overnight. Blocks sites where no protein has transferred.
12. Wash the blocked membrane in PBS 1X for 5 minutes.
13. Transfer the membrane into a solution of primary antibody (Rabbit anti-latex) in PBS. Dilution depends on titre of sera. Incubate for 1 hour or overnight.
14. Wash membrane 3X with 20 ml PBS.
15. Place membrane in 2^o antibody, (goat anti-rabbit, Promega). Dilute 1:5000. Incubate at least 1 hour. Secondary antibody is an AP enzyme conjugated Ab.
16. Wash membrane 3X with 20 ml PBS.
17. Make up developer solution, 10 mls of AP buffer, 33 ul of BCIP and 66 ul of NBT. Place membrane in solution and incubate until color develops in positive control lane. NBT/BCIP amounts are based upon Promega reagents. Color should develop in 15 minutes and no longer than 30 minutes.
18. Stop reaction by rinsing with water and air dry.  BCIP hydrolysis produces indigo ppt after oxidation with NBT.